Abstract
Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a ‘fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures.
Highlights
Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure
To test whether EY-CBS detects the formation of a helix, we chose two amino acid residues separated by 11 amino acids in the proposed fusion helix and mutated them to cysteines
To generate the EY-CBS reaction site, two amino acid residues separated by 11 amino acids, one from the ankyrin domain and the other from the protein A domain, were chosen and mutated to cysteines
Summary
Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Linking two protein components to form a predictable and rigid structure is a prerequisite for generating complex protein assemblies in a pre-designed fashion[1]. The resulting hybrids have significant structural flexibility and the relative orientation and distance between their two components is largely unpredictable This is the case even when the chemical cross linkers themselves have rigid structures since they are attached to flexible side chains such as cysteines or lysines. By testing various linker sequences they were able to find one that could form an a-helix and supported the formation of cage-like structures They applied a similar method to generate large and porous cube-like closed structures with high accuracy[17]. We propose a novel method for connecting the pre-existing a-helices of two proteins into a single extended helix using a chemical cross linker. It can be used to assemble protein components into asymmetrical and non-repeating structures
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